1. Field of the Invention
The present invention relates to an electrochemical test device suitable for determining the presence or concentration of chemical and biochemical components (analytes) in aqueous fluid samples and body fluids such as whole blood. Additionally, this invention relates to a method of using such test devices for determining the presence or concentration of an analyte and to processes for preparing such a test devices.
2. State of the Art
Medical studies have demonstrated that the incidence of serious complications resulting from diabetes, such as vision loss and kidney malfunction, can be significantly reduced by careful control of blood glucose levels. As a result, millions of diabetics use glucose testing devices daily to monitor their blood glucose concentrations. Additionally, a wide variety of other blood testing devices are used to determine the presence or concentration of other analytes, such as alcohol or cholesterol, in aqueous samples, such as blood.
Such blood testing devices typically employ either a dry chemistry reagent system or an electrochemical method to test for the analyte in the fluid sample. In recent years, electrochemical testing systems have become increasingly popular due to their small size and ease of use. Such electrochemical testing systems typically use electrochemistry to create an electrical signal which correlates to the concentration of the analyte in the aqueous sample.
Numerous electrochemical testing systems and related methods are known in the art. For example, European Patent Publication No. 0 255 291 B1, to Birch et al., describes methods and an apparatus for making electrochemical measurements, in particular but not exclusively for the purpose of carrying out microchemical testing on small liquid samples of biological, e.g. of clinical, origin.
European Patent Publication No. 0 351 891 B1, to Hill et al., teaches a method of making an electrochemical sensor by printing. The sensor is used to detect, measure or monitor a given dissolved substrate in a mixture of dissolved substrates, most specifically glucose in body fluid.
U.S. Pat. No. 5,391,250, to Cheney II et al., teaches a method of fabricating thin film electrochemical sensors for use in measuring subcutaneous or transdermal glucose. Fabrication of the sensors comprises placing a thin film base layer of insulating material onto a rigid substrate. Conductor elements for the sensors are formed on the base layer using contact mask photolithography and a thin film cover layer.
U.S. Pat. No. 5,437,999, to Diebold et al., teaches a method of fabricating thin film electrochemical devices which are suitable for biological applications using photolithography to define the electrode areas. The disclosures of each of the above patent specifications are incorporated herein by reference in their entirety.
An excellent reference on materials and process for fabricating electronic components is Charles A. Harper, Handbook of Materials and Processes for Electronics, 1984, Library of Congress card number 76-95803. It provides detail process information on thick film, thin film and photoresist processes.
Existing electrochemical testing systems, however, have certain limitations from the perspective of the end user or the manufacturer. For example, some electrochemical testing systems are difficult or costly to manufacture. As a result, such devices are too expensive to be used on a daily basis by, for example, diabetics. Other electrochemical testing systems are not sufficiently accurate to detect certain analytes at very low concentrations or to give reliable measurements of the analyte""s concentration. Additionally, many electrochemical devices are too large to be easily carried by those needing to test their blood on a regular basis throughout the day. Thus, a need exists for improved electrochemical test devices.
The present invention utilizes amorphous semiconductor materials and semiconductor and printed circuit board (PCB) manufacturing techniques to provide an electrochemical test device suitable for determining the presence or concentration of analytes in aqueous fluid samples. By using amorphous semiconductor materials and PCB manufacturing techniques, uniform electrochemical test devices having well-defined reproducible electrode areas can be manufactured economically.
In particular, the test devices of this invention have very uniform surface areas which reduces the variability of the electrochemical test. In this regard, it has been found that the surface area of the electrodes and the accuracy of applying the reagent are critical to producing an accurate test. If the surface area is not consistent from test to test then each of the test devices must be individually calibrated to insure accurate readings. The test devices of the present invention permit highly accurate electrochemical analyte measurements to be performed on very small aqueous fluid samples without the need for individual calibration of each test device. The present inventions provide for the accurate reproduction of the test devices by using controlled deposition methods, such as sputtering, and chemical machining methods to accurately form the geometries of consistent size and shape from device to device in continuous production. These devices can also be readily manufactured due to the lower cost and the flexible nature of the amorphous semiconductor materials which facilitates production by continuous roll processing versus the step and repeat printing methods currently employed. The ability to use continuous processing to fabricate the device, such as continuous processes utilizing continuous roll coating, continuous roll sputtering, continuous photolithography systems utilizing contact masks and flow through baths, results in high volume manufacturing capability an substantial cost reductions over the step and repeat processes. Additionally, the amorphous nature of the conductors electrodes and constructed and used according to this invention eliminates problems found in prior test devices which utilize conventional conductor and semiconductor materials, which are crystalline in nature or are noble metals and, as a result, require flat and rigid substrates to prevent cracking during manufacture, distribution or use.
Dry electrochemical test devices fall into two primary configurations. The first configuration utilizes two electrodes, i.e., a working electrode and a counter electrode. The second configuration utilizes three electrodes, i.e., a working electrode, a counter electrode and a reference electrode. The use of the reference electrode and a reference material provides a fixed reference for the test. The test devices of the present invention can be of either configuration.
Accordingly, in one of its aspects, the present invention provides an electrochemical test device for determining the presence or concentration of an analyte in an aqueous fluid sample, said electrochemical test device comprising:
(a) a non-conductive surface;
(b) a working electrode comprising an amorphous semiconductor material affixed to the non-conductive surface, said working electrode having an first electrode area, a first lead and a first contact pad;
(c) a counter electrode comprising an amorphous semiconductor material affixed to the non-conductive surface, said counter electrode having an second electrode area, a second lead and a second contact pad; and
(d) a reagent capable of reacting with the analyte to produce a measurable change in potential which can be correlated to the concentration of the analyte in the fluid sample, said reagent overlaying at least of portion of the first electrode area of the working electrode.
In another embodiment of this invention, the test device further comprises a reference electrode comprising an amorphous semiconductor material affixed to the non-conductive surface, said reference electrode having a third electrode area, a third lead, and a third contact pad, and wherein at least a portion of the third electrode area is overlaid with a reference material. Preferably, the reference material is a silver/silver chloride layer, a mercury/mercury chloride layer or a platinum/hydrogen material.
The non-conductive surface used in the test device of this invention can be any rigid or flexible material which has appropriate insulating and dielectric properties such as ceramics, polymeric board materials, flexible polymer sheets and the like.
Preferably, the non-conductive surface comprises a non-conductive coating affixed to one side of a flexible substrate comprising a metallic sheet material or a polymeric sheet material, such as polyester, polycarbonate and polyimide sheets or films. Preferred metallic sheets are metal foils which include aluminum, copper and stainless steel foil. Aluminum and stainless steel foil are particularly preferred.
The non-conductive coating used in the electrochemical test device is preferably an epoxy resin. The purpose of this coating is to provide a non-conductive barrier between the base material and the conductive layer and to improve the flatness of the surface morphology of the non-conductive surface on which the amorphous semiconductor electrodes are formed according to this invention. Better surface morphology of the non-conductive layer and the amorphous semiconductor electrodes provides improved accuracy of test results and consistency of performance.
Preferably, the amorphous semiconductor material used in this invention is amorphous silicon oxide. More preferably, the amorphous silicon oxide is doped with lithium, potassium, or a similar conducting ion to increased conductivity. Doping with lithium is particularly preferred. However, amorphous carbon, gold, silver or other conductor materials which do not interfere with the electrochemistry of the reagent system are also suitable. The amorphous semiconductor material can be applied by using various techniques including sputtering, evaporation, vapor phase deposition or other thin film deposition technique to form a conductive layer on the non-conductive surface, and technology can be used to form the electrodes. Thick film technologies can also be employed when using processes which control the application of the material and provide for uniform surface morphology. The surface texture of the amorphous semiconductor material is preferably less than 13 microinches or 0.33 microns. However, rougher textures can be used depending on the accuracy of the desired test device.
The reagent employed in the electrochemical test device is typically selected based on the analyte to be tested and the desired detection limits. The reagent preferably comprises an enzyme and a redox mediator. When the analyte to be detected or measured is glucose, the enzyme is preferably glucose oxidase and the redox mediator is potassium ferrocyanide.
In a preferred embodiment of this invention, the test device further comprises a blood separating membrane. The blood separating membrane separates whole blood samples into highly colored and relatively clear fluid portions before analysis. The blood separating membrane effectively blocks or filters red blood cells and allows essentially clear fluid to pass to the test electrodes. As a result, the analyte is measured in the clear fluid portion of the sample contacting the electrodes thereby substantially eliminating the red blood cells from the reaction and minimizing interference from the cells present in blood. This embodiment has the additional benefit of keeping the test site from drying out and thereby improves the performance of test devices designed for small sample sizes, such as in the 1 to 5 microlites range.
The electrochemical test device of the present invention is used to determine the presence or concentration of an analyte in an aqueous fluid sample. Accordingly, in one of its method aspects, the present invention provides a method for determining the presence or concentration of an analyte in an aqueous fluid sample, said method comprising:
(a) providing an electrochemical test device comprising: (i) a non-conductive surface; (ii) a working electrode comprising an amorphous semiconductor material affixed to the non-conductive surface, said working electrode having an first electrode area, a first lead and a first contact pad; (iii) a counter electrode comprising an amorphous semiconductor material affixed to the non-conductive surface, said counter electrode having a second electrode area, a second lead, and a second contact pad; and (iv) a reagent capable of reacting with the analyte to produce a measurable change in potential which can be correlated to the concentration of the analyte in the fluid sample, said reagent overlaying at least of portion of the first electrode area of the working electrode;
(b) inserting the electrochemical test device into a meter device;
(c) applying a sample of an aqueous fluid to the first electrode area of the working electrode;
(d) reading the meter device to determine the presence or concentration of the analyte in the fluid sample.
In another embodiment, the test device employed in this method further comprises a reference electrode comprising an amorphous semiconductor material affixed to the non-conductive surface, said reference electrode having a third electrode area, a third lead, and a third contact pad, and wherein at least a portion of the third electrode area is overlaid with a reference material. Preferably, the reference material is a silver/silver chloride layer, a mercury/mercury chloride layer or a platinum/hydrogen material. Silver/silver chloride is a particularly preferred reference material.
The non-conductive surface may be any rigid or flexible material having appropriate insulating and dielectric properties, as mentioned above. Preferably, the non-conductive surface is provided by affixing a non-conductive coating to one side of a substrate, which substrate is preferably a flexible metallic sheet material or a polymeric sheet material.
As discussed above, the present invention utilizes amorphous semiconductor materials and PCB manufacturing techniques to provide electrochemical test devices. This film or thick film methods and technologies can be used to create the amorphous semiconductor material conductive layers and electrodes according to this invention. Accordingly, in one of its process aspects, the present invention provides a process for preparing an electrochemical test device suitable for determining the presence or concentration of an analyte in an aqueous fluid sample, said process comprising the steps of:
(a) providing a non-conductive surface;
(b) depositing an amorphous semiconductor material on said surface to form a conductive layer;
(c) chemically machining the conductive layer to form a working electrode comprising a first electrode having a first electrode area, a first lead and a first contact pad, and to form a counter electrode comprising a second electrode having a second electrode area, a second lead and a second contact pad;
(d) applying a reagent to at least a portion of the first electrode area of the working electrode, said reagent being capable of reacting with an analyte in an aqueous fluid sample to produce a measurable change in potential which can be correlated to the concentration of the analyte in the fluid sample.
In another embodiment, step (c) of this process further comprises forming a reference electrode comprising a third electrode having a third electrode area, a third lead and a third contact pad.
Preferably, the non-conductive surface is provided by affixing a non-conductive coating to one side of a substrate, which substrate is preferably a flexible metallic sheet material or a polymeric sheet material. Accordingly, in a preferred embodiment, step (a) above comprises the steps of:
(f) providing a flexible substrate; and
(g) applying a non-conductive coating to the substrate to form a non-conductive surface.
In another preferred embodiment, step (c) above comprises the steps of:
(h) applying a photoresist to the conductive layer to form a first photoresist layer;
(i) positioning a first developer mask on the first photoresist layer;
(j) exposing the unmasked first photoresist layer to ultraviolet light to form a first patterned photoresist area;
(k) removing the first developer mask;
(l) removing the first photoresist layer not exposed to ultraviolet light with a developer to form a first exposed conductive layer;
(m) contacting the first exposed conductive layer with a chemical etchant to remove the first exposed conductive layer; and
(n) removing the first patterned photoresist area with a solvent to form a second exposed conductive layer, said second exposed conductive area comprising (i) a working electrode comprising a first electrode having a first electrode area, a first lead and a first contact pad, (ii) a counter electrode comprising a second electrode having a second electrode area, a second lead and a second contact pad, and optionally (iii) a reference electrode comprising a third electrode having a third electrode area, a third lead and a third contact pad.
In further preferred embodiment, step (c) above fturther comprises the steps of:
(o) applying a photoresist to the second exposed conductive layer to form a second photoresist layer;
(p) positioning a second developer mask on the second photoresist so that the second photoresist layer covering the third electrode area is masked;
(q) exposing the unmasked second photoresist layer to ultraviolet light to form a second patterned photoresist layer;
(r) removing the second developer mask;
(s) removing the second photoresist layer not exposed to ultraviolet light with a developer to expose the third electrode area;
(t) applying a reference material the third electrode area;
(u) removing the second patterned photoresist layer with a solvent.
Preferably, the process employed to prepare the test devices of this invention is a continuous process. The ability to use continuous processing to fabricate the test devices, such as a continuous process utilizing continuous roll coating, continuous roll sputtering, continuous photolithography systems utilizing contact masks and flow through baths, results in high volume manufacturing capability and substantial cost reductions over the step and repeat processes.